Glia and endothelial cells play a central role in the homeostatic regulation of the extracellular environment of the nervous system. The ionic composition and overall extracellular volume is actively regulated by these cells. In many pathological conditions homeostatic balance is lost, leading to swelling of the extracellular space, neuronal dysfunction, and apoptosis. Despite the severe impact of glial dysfunction, much remains to the learned about the molecular mechanisms that govern this critical form of physiological regulation. The focus of the study is a signaling cascade mediated by the Drosophila ser/thr kinase Fray. Fray regulates the activity of its downstream effector, the cation-chloride cotransporter NCC69, in glial cells of the blood-nerve barrier. This cascade is involved in the regulation of extracellular volume in peripheral nerves: nerves defective in this signaling cascade have extensive swelling. Such swelling also occurs in hyperactie mutants that have increased neuronal electrical activity. We have found extensive molecular and functional homologies between the Drosophila proteins and their human counterparts, SPAK and NKCC1. There is good evidence that dysfunction of NKCC1 in mammals is associated with stroke, and leads to edema in the nervous system. In this project we will examine the molecular cascade in detail, with the goal of identifying novel components, and to better understand how neural activity affects glial function. In specific aim 1 we use a genetic approaches to characterize the molecular elements that are involved in activity-dependent swelling in peripheral nerves. In the second specific aim we will use a knock-down RNAi screen to identify a substantial number of previously unidentified molecular players that function at the blood-nerve barrier to regulate the extracellular environment of the nervous system. The use of a genetic model system with extensive mammalian homology is a powerful strategy to rapidly identify the key molecular components involved in glial homeostasis. With their identification, this R21 proposal will set the stage for follow-on studies to better resolve how glia regulate their extracellular environment.